Maspin, Maspin Derivatives, and Maspin Mimetics for Reducing ROS, Inflammation, and Skin Aging

ABSTRACT

The present invention provides compositions, methods, and systems for treating inflammatory conditions (e.g., by inhibiting reactive oxygen species) in or on a subject with maspin, maspin derivatives, or maspin mimetics. In some embodiments, such agents are applied to the skin of a subject (e.g., to reduce skin aging).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/945,882, filed Nov. 19, 2015, which claims priority to U.S.Provisional Application Ser. No. 62/082,456, filed Nov. 20, 2014, whichis herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under CA079736 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention provides compositions, methods, and systems fortreating inflammatory conditions (e.g., by inhibiting reactive oxygenspecies) in or on a subject with maspin, maspin derivatives, or maspinmimetics. In some embodiments, such agents are applied to the skin of asubject (e.g., to reduce skin aging).

BACKGROUND

Oxidative stress, a hallmark of many tumors, inflammatory response, andcell aging, is caused by an imbalance between the generation of reactiveoxygen species (ROS) and cells' ability to clear oxidants. Processesassociated with proliferation, apoptosis and senescence may be theresult of activation of signaling pathways in response to intracellularchanges in ROS. Thus, excessive production and inadequacy in a normalcell's antioxidant defense system can cause the cell to experienceoxidative stress.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods, and systems fortreating inflammatory conditions (e.g., by inhibiting reactive oxygenspecies) in or on a subject with maspin, maspin derivatives, or maspinmimetics. In some embodiments, such agents are applied to the skin of asubject (e.g., to reduce skin aging) or internally to treat cancer.

In some embodiments, provided herein are methods of treating aninflammatory condition, or cancer, comprising: administering to asubject a composition comprising maspin, a maspin derivative, or amaspin mimetic. In certain embodiments, the administering is to the skinof a subject.

In certain embodiments, provided herein are compositions comprising: i)a human skin moisturizing lotion, and ii) maspin, a maspin derivative,or a maspin mimetic.

In some embodiments, provided herein are methods of treating aninflammatory condition and/or skin aging comprising: administering to asubject (e.g., who has symptoms of an inflammatory condition and/or skinaging) a composition (e.g., a pharmaceutical composition) comprisingmaspin, a maspin derivative, or a maspin mimetic.

In certain embodiments, the administering is to the skin of the subject(e.g., by applying a spray, lotion, cream, gel, ointment, or applying askin patch). In particular embodiments, the composition furthercomprises skin lotion, cream, gel, or an ointment, a spray, or ispresent in a skin patch. In some embodiments, the skin of the subject isaging skin (e.g., the subject is a human that is over 50 . . . 60 . . .70 . . . 80 . . . or 90). In other embodiments, the skin of the subjectis wrinkled. In additional embodiments, the skin has been damaged due toexposure to ultraviolet light.

In certain embodiments, the present disclosure provides methods oftreating skin, skin conditions, and/or aging skin. In some embodiments,the methods are directed to improving skin appearance. For example, acomposition such as described herein can be applied to the skin of asubject on or near areas in which an improvement in skin appearance isdesirable, such as the face, or portions thereof, near or around theeyes and/or mouth, or the arms, legs, back, stomach, etc. In someembodiments, the methods are directed to reducing or preventing theappearance of skin wrinkles. In some embodiments, the methods aredirected to improving the appearance of skin that has been damaged dueto exposure to ultraviolet light (e.g., sunburned skin). In furtherembodiments, the methods are directed to improving the appearance ofaging skin (e.g., by reducing or preventing the appearance of wrinklesand/or improving the elasticity of the aging skin).

In particular embodiments, the maspin derivative comprises an amino acidsequence selected from SEQ ID NOs:1-4. In other embodiments, the maspinderivative comprises an amino acid sequence selected from SEQ IDNOs:5-7. In some embodiments, the maspin derivative comprises the aminoacid sequence in SEQ ID NO:8. In additional embodiments, the maspinderivative further comprises a peptide amphiphile. In furtherembodiments, the maspin derivative comprises (or consists of, orconsists essentially of) an amino acid sequence selected from SEQ IDNOs:9-12. In additional embodiments, the maspin is full length, or atleast 97%, 98%, or 99% of, full-length human maspin or its homologs inother species (e.g., rat, mouse, dog, etc.). In certain embodiments, themaspin is full-length human maspin minus 3-5 amino acids at the N, C orboth termini.

In other embodiments, the subject has, or is suspected of having,symptoms of an inflammatory condition selected from the group consistingof: benign prostate hyperplasia (BPH), inflammatory bowel disease (IBD),ROS-induced obesity, diabetes, and atherosclerosis. In particularembodiments, the administering reduces or eliminates at least one of thesymptoms of the inflammatory condition.

In some embodiments, provided herein are compositions comprising: i) ahuman skin moisturizing lotion, cream, gel, ointment, and ii) maspin, amaspin derivative, or a maspin mimetic. In other embodiments, providedherein is a skin patch device, wherein the skin patch comprises maspin,a maspin derivative, or a maspin mimetic (e.g., configured to bedelivered over time to the skin of a subject wearing the patch).

In certain embodiments, provided herein are systems and kits comprising:i) a human skin moisturizing lotion, and ii) maspin, a maspinderivative, or a maspin mimetic. In some embodiments, the maspinderivative comprise an amino acid sequence selected from SEQ IDNOs:1-13.

DESCRIPTION OF THE FIGURES

FIGS. 1A-E. Total intracellular ROS levels in primary mammary epithelialcells isolated from WT and heterozygous maspin KO mice and maspinsilenced immortalized human mammary epithelial MCF-10A. Totalintracellular ROS levels were measured by flow cytometric analysis usingCarboxy-DCFH-DA as a fluorogenic substrate. (A) Western blot analysis ofmaspin expression in primary mammary epithelial cells isolated from WTand heterozygous maspin KO mice. Equal amount of proteins (25 μg/lane)were loaded and immunobloted for maspin and actin (a loading control).(B) Constitutive levels of ROS in primary mammary epithelial cellsisolated from WT and heterozygous maspin KO mice (n=3). (C) ROS level inprimary mammary epithelial cells isolated from WT and heterozygousmaspin KO mice (n=3) after treatment with H2O2 (250 μM) for 3 hrs. (D)Western blot analysis of maspin expression in immortalized human mammaryepithelial MCF-10A and MCF-10A229 cells. (E) ROS level in immortalizedhuman mammary epithelial MCF-10A and MCF-10A229 (maspin silenced) cellsafter treatment with H2O2 (250 μM) for 3 hrs. Data shown are mean+SD ofthree independent experiments. Asterisks indicate significance accordingto t-test (two-tailed); *p<0.05, **p<0.01, ***p<0.001

FIGS. 2A-E. Maspin overexpressing tumor cells are resistant to oxidativestress. (A) Western blot analysis of maspin expression in TM40DNeo andTM40DMp cells. Equal amount of proteins (25m/lane) were loaded andimmunobloted for maspin and actin (a loading control). (B) ROS level inTM40DNeo and TM40DMp cells after treating with H2O2 (250 μM) for 3 hrs.(C) ROS level in TM40DNeo and TM40DMp cells after treating withantimycin-A (100 μM) for 1 hr. (D) Schematic representation of oxidationof hydroethidine (HE) and formation of various oxidation products.2-hydroxyethidium (2-OH-E+) is the primary product of the reaction of HEand superoxide (O2°-) and other products like E+ and dimers of HE(HE-HE, HE-E+, E+- E+) are indicators of one electron oxidants. (E)Levels of superoxide (2-OH-E+) in TM40DNeo and TM40DMp cells aftertreating with STS (1 μM) for 3 hrs as measured by HPLC-MS. The resultsare shown as area under peak per mg of protein. Data shown are mean+SDof three independent experiments. Asterisks indicate significanceaccording to t-test (two-tailed); *p<0.05, ***p<0.001.

FIGS. 3A-D. Importance of cysteine residues and role in oxidativestress. (A) Three dimensional structure of maspin highlighting threecysteine residues marked in green were mutated to serine at position183, 205 and 323. (B) Western blot analysis of maspin expression inTM40DNeo, TM40DMp, TM40DT and other maspin mutant cells. Equal amount ofproteins (25 μg/lane) were loaded and immunobloted for maspin and actin(a loading control). (C) Comparison of total intracellular ROS levels invarious TM40D cells treated with H2O2 (250 μM) for 3 hrs as determinedby flow cytometric analysis. (D) Constitutive levels of GSH/GSSG ratioin TM40DNeo, TM40DMp and TM40DT cells. Data shown are mean+SD of threeindependent experiments. Asterisks indicate significance according tot-test (two-tailed); **p<0.01, ***p<0.001 compared with TM40DMp; #p<0.05 compared with TM40DT.

FIGS. 4A-D. Triple mutated maspin is biologically active and evidencefor oxidation of cysteine to sulfenic acid. (A) Mutated maspin retainsthe ability to enhance cell adhesion to extracellular matrix (ECM).Increased adhesion of TM40DNeo cells to matrigel ECM was observed whencells were pretreated with wild type and triple mutated recombinantmaspin (100 nM) as compared to its GST control. (B) Biochemistry ofoxidation of thiol group [—SH] and capturing of sulfenic acid [—SOH]using dimedone. Protein thiols [—SH], which are susceptible to oxidationby ROS, generate sulfenic acid [—SOH] and largely irreversible sulfinicacid [—SO2H] and sulfonic acid [—SO3H]. Sulfenic acid can be labeled bydimedone or dimedone based chemicals. (C) Equal amounts (62.5 ng) of WTand Triple mutated maspin were oxidized with H2O2 (10 mM) for 1 minutein presence of dimedone (10 mM) and oxidation of cysteine residues wasdetected by Western blot analysis using antibody, which specificallyrecognize dimedone derivatized cysteine sulfenic acid residues (D) Cellswere treated with H2O2 (250 μM) for 3 hrs and then lysed in presence orabsence of Dimedone (10 mM). Maspin was immunoprecipitated and thenimmunobloted with maspin antibody and antibody, which specificallyrecognizes dimedone derivatized cysteine sulfenic acid residues. Datashown are mean+SD of three independent experiments. Asterisks indicatesignificance according to t-test (two-tailed); **p<0.01, ***p<0.001.

FIGS. 5A-E. Cysteine mutated cells have increased cellular proliferationand ERK1/2 activation. (A) Number of colonies formed in vitro using asoft agar colony formation assay by different cell lines. (B) Comparisonof cellular proliferation pattern as determined by MTT assay in threedifferent cell lines. (C) Treatment with anti-oxidant (i.e. NAC)significantly decreases the proliferation in TM40DNeo and TM40DT cells.(D) Comparison of pERK1/2 levels in TM40DNeo and TM40DT cells comparedwith TM40DMp cells. (E) Effect of NAC treatment on pERK1/2 levels inTM40DNeo, TM40DMp and TM40DT cell. Representative Western blots areshown above the bar diagrams and each lane corresponds to respectivebars. Data shown are mean+SD of three independent experiments. Asterisksindicate significance according to t-test (two-tailed); **p<0.01,***p<0.001

FIG. 6. Maspin interacts with Trx-1 in redox regulation. FLAG-Trx-1 wasimmunoprecipitated from cell lysates of MCF-7-Flag-Trx-C35S cells andanalyzed for co-immunoprecipitation of maspin. IgG served as negativecontrol. Input corresponds to the cell lysates beforeImmunoprecipitation. Top: immunoblot with an anti-maspin rabbitpolyclonal antibody; bottom: immunoblot with an anti-FLAG antibody.LC=Light chain, HC=Heavy Chain.

DETAILED DESCRIPTION

The present invention provides compositions, methods, and systems fortreating inflammatory conditions (e.g., by inhibiting reactive oxygenspecies) in or on a subject with maspin, maspin derivatives, or maspinmimetics. In some embodiments, such agents are applied to the skin of asubject (e.g., to reduce skin aging).

Maspin, a member of the serine protease inhibitor (serpin) superfamily,displays tumor suppressing activity by controlling cell migration,proliferation, apoptosis and adhesion. Provided herein is evidence thatmaspin acts as a reactive oxygen species (ROS) scavenger throughoxidation of three structurally exposed cysteine thiols to sulfenicacid. Ablation of these cysteine residues in maspin results in asignificant increase in total ROS production in TM40D mouse mammarycells. Also, the cells containing triple cysteine mutant of maspin showelevated ERK1/2 activity, a downstream target of ROS, and enhancedproliferation and colony formation. These findings establish a novelmechanism by which maspin utilizes its cysteine thiols to inhibitoxidative stress and cell growth. In addition, Maspin's anti-ROSproperty may be used against inflammation-induced responses and skinaging. Inflammation can be induced due to excessive free radicals andoxidative stress. Inhibiting ROS using maspin may block inflammatoryresponse. Skin aging is well known to be regulated by an imbalance incellular ROS of skin epithelial cells. Topical application ofrecombinant maspin may inhibit ROS level and block skin aging process.

Maspin is endogenously expressed in epithelial cells and its expressioncan be up-regulated by a range of factors. Maspin mimetic peptides canbe designed to mimick whole protein effect against ROS, making it easierto produce for treatment. Recombinant maspin or its derivative includingmimetic peptides may be applied topically on skin to inhibit cellularROS, while excessive skin cell ROS is critical for skin cell aging.Thus, maspin and its derivative may be used as an anti-aging product forskin care.

Maspin has eight cysteine residues, which prompted an exploration ofcysteine-targeted oxidation of this multifaceted protein in theregulation of ROS metabolism. It was found that only three cysteineresidues, located at positions C183, C205, and C323 are structurallyfully exposed. Given the anti-oxidant capacity of the cysteine thiolgroup, it was hypothesized and proved that these exposed cysteineresidues in maspin act as a potent scavenger/quencher of ROS. Maspinoverexpressing cells are more resistant to oxidative stress and thisproperty is attributed to the cysteine residues in maspin. Recombinantmaspin serves to inhibit ROS in the cell-free system.

Maspin acts as a reactive oxygen species (ROS) scavenger. Threestructurally exposed cysteine thiols in maspin are responsible for thisanti-ROS function. These bases have been identified as C183, C205, andC323. Their relative position in three dimension structure is shown inFIG. 3A. In addition, other cysteine residues (C20, C287, and C373) inmaspin are identified to play a role in ROS inhibition, by formingintramolecular disulfide bond and interacting with Trx1.

In certain embodiments, a maspin derivative is employed in the methods,kits, and compositions herein. Exemplary maspin derivative sequences areprovided in Table 1 below. Such sequence, alone, or with additionalmaspin or non-maspin amino acids at either end (or other molecules suchas peptide amphiphiles) may be employed.

TABLE 1 A. Maspin truncated peptides 1. amino acids 190-202:(SEQ ID NO: 1) TDTKPVQMMNMEA; 2. s2C 265-274: (SEQ ID NO: 2)ANAKVKLSIPK; 3.  (SEQ ID NO: 3) NPSTMANAKVKLSIPK; 4. (SEQ ID NO: 4)TDTKPVQMMNMEATFCMGNIDSI: 181-202 and 190-211;B. Maspin derivative peptides 5. (SEQ ID NO: 5) STANAKVKLSIP; 6.(SEQ ID NO: 6) TANAEVKLSIPK; 7. (Seq ID NO: 7) STENAKVKLSIP;C. Maspin G-helix peptide (effective componentfor the maspin nanopeptide MMPA) 8. (SEQ ID NO: 8) EDESTGLEKIEKQLN;D. Additional maspin cysteine residues for Trx1 interaction 9.(SEQ ID NO: 9) ALVDLFKQLCEKEPG-XXXX-SCFKGFFI; 10. (SEQ ID NO: 10)ALVDLFKQLCEPGACFKGFFI; 11. (SEQ ID NO: 11)ALVDLFKQLCEKEPG-XXXX-ACLENLGL; 12. (SEQ ID NO: 12)ALVDLFKQLCEPGACLENLGL.In the above, “X” may be any amino acid residue. Also, the sequence,such as SEQ ID NO:8, may be part of a peptide-amphiphile as described,for example, in Pat. Pub. US20140256635, which is herein incorporated byreference in its entirety, particular for MMPA constructs. In certainembodiments, one or more additional human maspin amino acids (or otheramino acids) are added to the ends of the sequences shown in Table 1(e.g., 5-25 or 25-50 additional amino acids from human maspin areadded). In certain embodiments, the maspin employed is full length or atleast 99% of full-length human maspin. A full length human maspin isfound at Genbank accession number AAA18957, which provides the fullhuman protein. This full length protein is also shown below:

(SEQ ID NO: 13)   1mdalqlansa favdlfkqlc ekeplgnvlf spiclstsls laqvgakgdt aneigqvlhf  61envkdipfgf qtvtsdvnkl ssfyslklik rlyvdkslnl stefisstkr pyakeletvd 121fkdkleetkg qinnsikdlt dghfenilad nsvndqtkil vvnaayfvgk wmkkfpeset 181kecpfrinkt dtkpvqmmnm eatfcmgnid sinckiielp fqnkhlsmfi llpkdvedes 241tglekiekql nseslsqwtn pstmanakvk lsipkfkvek midpkaclen lglkhifsed 301tsdfsgmset kgvalsnvih kvcleitedg gdsievpgar ilqhkdelna dhpfiyiirh 361nktrniiffg kfcsp.Maspin and maspin derives from other, non-human species may also beemployed. For example, maspin from mouse (AAB06042), rat (AAB06043),horse, cow, dog, cat, etc.

In certain embodiments, the compositions (comprising the maspin, maspinderivatives or mimetics) are formulated in a format selected from thegroup consisting of a cream, a lotion, a spray, an ointment, a gel, apowdered mask, a paste, a cleanser, and a foundation. In otherembodiments, such compositions are present in a patch. In particularembodiments, the compositions may include one or more additives selectedfrom the group consisting of: a perfume, colorant, thickening agent,vegetable oil, emulsifier, solvent, pH adjusting agent, antisepticagent, preservative, vitamin, sun-block, surfactants and combinationsthereof. Various physical sunscreen agents such as titanium dioxide,silicone-treated titanium dioxide, zinc oxide, ferrous oxide, ferricchloride, talc, chromium oxide, or cobalt oxides may be included.Alternatively or in addition, a chemical sunscreen agent such aspara-amino benzoic acid, esters of para-amino benzoic acid, salicylates,cinnamates, benzophenones, dihydroxyacetone, parsol 1789, or melanin maybe included.

In certain embodiments, the compositions disclosed herein are used totreat a subject who has, or is suspected of having, symptoms of aninflammatory condition, such as, but not limited to: benign prostatehyperplasia (BPH), inflammatory bowel disease (IBD), ROS-inducedobesity, diabetes, and atherosclerosis. In particular embodiments,administering the composition to the subject (e.g., intravenously)reduces or eliminates at least one of the symptoms of the inflammatorycondition.

Compositions for such in vivo administration to a subject can beformulated in pharmaceutical compositions. Such pharmaceuticalcomposition, besides containing maspin, maspin derivatives or mimetics,may contain additional agents. It is not intended that the presentinvention be limited by the particular nature of the pharmaceuticalpreparation. For example, such compositions can be provided togetherwith physiologically tolerable liquid, gel or solid carriers, diluents,adjuvants and excipients. These therapeutic preparations can beadministered to mammals for veterinary use, such as with domesticanimals, and clinical use in humans in a manner similar to othertherapeutic agents. In general, the dosage required for therapeuticefficacy will vary according to the type of use and mode ofadministration, as well as the particularized requirements of individualhosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Oral formulations usually will includesuch normally employed additives such as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%. The compositions are also prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The compositions of the present invention may be mixed withdiluents or excipients which are physiological tolerable and compatible.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

In some embodiments, the pharmaceutical composition are administered toa subject, or applied to the skin of a subject, in an amount within therange of from about 0.01 mg/kg to about 100 mg/kg (maspin or maspinderivative per kg of patient), although higher and lower doses may beutilized. For example, in some embodiments, between about 100-200,200-400, 400-800, or 800-1000 mg/kg or more of maspin or maspinderivative is administered or applied.

Anti-ROS reagents have exciting potential in cancer therapy as astrategy to control tumor growth. Because maspin is expressedendogenously in the epithelial cells, it is possible one can upregulatemaspin to enhance cell's ability against ROS thereby inhibiting cancerinitiation. This property may be used as a novel cancer therapy. Bothinflammatory response and aging are influenced by ROS. Inhibiting ROScan block inflammatory responses and aging. Maspin's anti-ROS propertymay be us used as a topically applied reagent against skin aging orother inflammatory diseases. The following are inhibitors againstoxidative stress currently under development: BG 12—Biogen Phase IIIdrug—anti-Ros agent; and NAC—Medicinal Nutraceutics—N-Acetyl Cysteine(NAC)—Capsules currently in the market.

Examples Example 1 Use of Maspin as an Anti-ROS Scavenger Against CellProliferation, Inflammation, and Aging Experimental Procedures

Plasmid constructs and cell culture—Maspin X-ray crystal structure wasreported by Law et al. (16) and Al-Ayyoubi et al. (17). Molsoft ICM-proVersion 3.48 was used to analyze the X-ray structure of human maspin(PDB ID: 1xu8), as reported by Law et al (16) to analyze the presence ofexposed cysteine residues, which can serve as site for oxidation.QuikChange II site-directed mutagenesis kit (Stratagene) was used tomutate cysteine residues to serine residues in pEF-IRES-neo-h.maspinusing the specific mutagenic primers (Table-S1). Mutations in theplasmid were confirmed by DNA sequencing.

Murine mammary tumor TM40D cells were used and maintained as describedpreviously (18). TM40D cells were transfected with pEF-IRES-neo-h.maspinwild type (Mp), triple mutation (T) or control vector alone (Neo) byEffectent reagent (Qiagen). The stable transfectants were selected withG418 medium (600 μg/ml) for 14 days and expression of maspin wasconfirmed by Western blot analysis. The GST fusion proteins[GST-h.maspin (GST-MpWT) and GST-h.maspin triple mutation (GST-MpT)]were induced by IPTG (1 mM), and purified using glutathione agarose(Sigma). Thrombin was used to cleave the maspin from the agarose beads.The size and purity of proteins were confirmed by SDS-PAGE and Westernblot analysis.

Human mammary tumor and immortalized epithelial cells (MCF-7, MCF-10A,MCF-10A229) were maintained as described previously (19). In a previousstudy, it was shown that maspin homozygous knockout mice (KO) areembryonically lethal (20), therefore, mouse primary mammary epithelialcells from wildtype (WT) were isolated and maspin heterozygous knockoutmice as described earlier (21).

Western Blot Analysis—Cell Lysates were Prepared in RIPA Buffer withProtease Cocktail Inhibitor (Thermo Scientific).

Cellular debris was cleared from lysates by centrifugation and proteinconcentration was determined by the BCA Protein Assay (Pierce). Sampleswere separated on 10% SDS-PAGE, transferred to a PVDF membrane (GEHealthcare) and blotted with rabbit anti-maspin AbS4A antibody (3) andanti-actin antibody (Cat No. A2066. Sigma). HRP labeled goat anti-rabbitpolyclonal antibody was used as a secondary antibody and proteins werevisualized with enhanced chemiluminescence substrate (Pierce).

Quantification of Reactive Oxygen Species (ROS)—Flurogenic substratecarboxy-2′, 7′-dicholorodi hydrofluorescein diacetate (DCFH-DA) was usedto detect intracellular ROS (22). Briefly, 10⁶ cells in 6-well platewere plated and incubated at 37° C. overnight. Next day, the culturemedium was discarded and cells were washed twice with PBS followed byincubation with 20 μM Carboxy-DCFH-DA at 37° C. for 30 minutes in serumfree medium. Cells that were either untreated or treated with differentROS inducers at the indicated final concentrations were incubated at 37°C. for indicated time periods. At the end of the exposure period, cellsupernatants were discarded; cells were washed with PBS and harvestedusing Trypsin/EDTA. Cells were transferred to FACS tubes and 20,000events were analyzed using a BC Epics XL Analyzer with an excitationwavelength of 488 nm and an emission wavelength of 525 nm. Results weredepicted as fold change in fluorescent intensity.

Measurement of Superoxide (O2°-) levels—To measure superoxide levels inthe cell culture, we used2,7-Diamino-10-ethyl-9-phenyl-9,10-dihydrophenanthridine,3,8-Diamino-5,6-dihydro-5-ethyl-6-phenylphenanthridine hydroethidine(HE) a fluorogenic probe, which is widely used to detect superoxidelevels (23). TM40DNeo and TM40DMp cells were treated with staurosporine(STS; 1 μM) for 3 hrs and incubated with 10 μM HE for 1 hr. Treatingcultured cells with STS is known to induce a rapid and prolongedincrease in ROS (24,25). Cells were washed twice with cold PBS, scrapedand kept for 30 minutes on ice. Cell suspension was centrifuged at1000×g for 5 minutes at 40 C. Pellets were either stored at −800 C orimmediately processed for HPLC analysis. Cells were lysed using 0.1%Triton X-100 in DPBS. Cell lysates were mixed with an equal amount of0.2 M solution of HClO4 in MeOH, and left undisturbed for 2 hrs to allowprotein precipitation. Samples were centrifuged for 30 minutes at 20,000g at 40 C and 100 μl of supernatant was mixed with an equal amount of 1M solution of potassium phosphate buffer (pH 2.6). After, centrifugingat 20,000 g for 15 minutes at 40 C, sample were analyzed by HPLC-MS.Instrumental setup for the analysis of HE and its oxidation products byHPLC-MS is depicted in Table-S2. HPLC peak areas were normalized toprotein concentration.

Cell-extracellular matrix (ECM) adhesion assay—Adhesion of mouse mammaryTM40DNeo cells to extracellular matrix matrigel (BD Biosciences) wasused to determine the biological activity of recombinant proteins.Adhesion assay was performed as described by Corbett et al (26).Briefly, cells were suspended in medium containing 100 nM of maspin(GST.MpWT and GST.MpT) or GST, and then cultured on matrigel coated96-well plate at a density of 5×104 cells/well in triplicates for 4 hrs.The plates were washed twice with PBS and incubated with 50 μl ofhexosaminidase substrate (3.75 mM 4-NitrophenylN-acetyl-β-D-glucosaminide, 0.25% Triton X-100, 0.05 M citrate buffer,pH 5) for 1.5 hrs at 370 C. After incubation 75 μl of development buffer(5 mM EDTA, 50 mM glycine, pH 10.4) was added and the readings wererecorded at 405 nm.

Measurement of GSH/GSSG ratios—A luminescence-based system was used fordetection and quantification of GSH/GSSG ratios in cultured cells (CatNo. V6611, Promega).

Detection of protein sulfenic acid modifications—Detection of proteinsulfenic acid in vitro was done as described previously (14,15,27).Briefly, equal amounts (62.5 ng) of thrombin cleaved WT (MpWT) andtriple mutated (MpT) recombinant maspin proteins were oxidized with H2O2(1 mM) for 1 minute in the presence of dimedone (10 mM). The oxidationof cysteine residues was detected by Western blot analysis using anantibody (Millipore; Cat No. 07-2139) that specifically recognizesdimedone derivatized cysteine sulfenic acid residues.

Trapping of sulfenic acid in cells (Immunoprecipitation)—Cells werefirst treated with H2O2 (250 μM) for 3 hrs and then lysed in lysisbuffer: 100 mM Tris (pH 7.4), 1% Triton X-100, protease inhibitormixture (Thermo Scientific) with or without dimedone (10 mM) for 45minutes before the addition of N-ethylmaleimide to a final concentrationof 100 mM for another 10 minutes. Cellular debris was cleared fromlysates by centrifugation, and protein concentration was determined bythe BCA Protein Assay (Pierce). Whole cell extracts (1 mg) wereincubated overnight (constant rocking) with 0.5 μg of rabbit anti-maspinAbS4A antibody (3) or control rabbit IgG at 4° C. ProteinA-Sepharose-coupled beads (Amersham Biosciences) were added andincubated for 1 hr at 4° C. under constant agitation. Beads werecentrifuged, washed briefly with ice-cold lysis buffer, and finallyincubated with elution buffer for 15 minutes at room temperature.Samples were mixed with non-reducing buffer (5×) and separated onSDS-PAGE gels, transferred to a PVDF membrane (GE Healthcare), andprobed for maspin, cysteine sulfenic acid and IgG. Appropriate secondaryantibodies were added, and proteins were visualized with enhancedchemiluminescence substrate (Pierce). Anti-Cysteine Sulfenic Acid wasobtained from Millipore (Cat No. 07-2139), which recognizes proteinscontaining the dimedone-bound cysteine sulfenic acid.

Soft agar colony formation assay—As described previously (28), 6-welldishes were plated with bottom agar (0.7% agarose) for 30 minutes. Cellswere mixed with the top agar (0.3% agarose) at a concentration of 5×104cells per well and allowed to solidify. The cells were fed every 5th daywith media and grown at 37° C. for 3 weeks, cells stained for 1 hr with0.05% crystal violet and the numbers of purple colored colonies werecounted as 10 fields per well at 40× using Image J software with a cutoff range of 20-5000 pixels.

MTT in vitro cell proliferation assay—Cells were seeded at 10³cells/well in 96-well plate and allowed to grow at 37° C. with 5% CO2.At each time point MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide) reagent (5 mg/ml) was added in a volume of10 μl per well and incubated at 37° C. with 5% CO2 for 3 hrs. The mediawas aspirated and 100 μl of DMSO was added and mixed until a uniformpurple color was formed. The cell samples were measured using a platereader at 570 nm. Assays were performed in triplicates.

ERK1/2 phosphorylation—Protein lysates (30m) were separated on 12%SDS-PAGE and phospho-plus kit was used to determine the ERK1/2phosphorylation (Cat No. 9100, Cell Signaling Technology).

Statistical Analysis—All the experiments were carried out three times oras stated. Quantification of Western Blots was performed using ScionImage. Statistical analysis (two tailed t-test) was based on a minimumof three replicates using Prism statistical software. The differenceswere considered significant if p<0.05.

Results

Status of intracellular ROS levels in primary mouse mammary epithelialcells isolated from wildtype (WT) and heterozygous maspin knockout (KO)mice and maspin silenced immortalized human mammary epithelial MCF-10Acells. In order to understand the role of maspin in oxidative stress, weisolated primary mammary epithelial cells from WT and maspinheterozygous KO mice and measured their ROS levels. FIG. 1A depicts theWestern blot analysis for maspin expression in cells isolated from WTand maspin heterozygous KO mice. We observed that the cells isolatedfrom maspin KO mice had significantly higher intracellular ROS levels ascompared with WT mice (p<0.001; FIG. 1B). We also observed a significantincrease in ROS production in KO cells as compared with WT cells whenthey were pre-treated with H2O2 for 3 hrs (p<0.05; FIG. 1C). To furtheranalyze the importance of maspin in ROS production, we usedmaspin-silenced MCF-10A cells (MCF-10A229), which as confirmed byWestern blot analysis, showed attenuated levels of maspin (FIG. 1D).MCF-10A cells displayed significantly lower levels of ROS as comparedwith the maspin-silenced cells MCF-10A229 cells (p<0.01; FIG. 1E).Collectively, these results indicate that presence of maspin in primaryand in immortalized mammary epithelial cells make them more efficient toquench ROS and therefore, indicate the importance of maspin in ROSregulation.

Maspin overexpressing cells are resistant to oxidative stress—Todirectly target the importance of maspin in oxidative stress, TM40Dmouse mammary epithelial cell line that expresses low levels of maspin,was used as a cellular model (FIG. 2A). Maspin was over expressed inTM40D cells (FIG. 2A) and total intracellular ROS levels were measured.Our results demonstrated that the exposure for 3 hrs to 250 μM H2O2 wassufficient to induce significant increase in intracellular ROS inTM40DNeo cells as compared to maspin overexpressing TM40DMp cells(p<0.001; FIG. 2B).

To determine the physiological relevance or the effect of endogenous ROSon mammary epithelial cells, we used antimycin-A, a compound thatinhibits electron transport at complex III of the mitochondrialrespiratory chain, thereby, inducing the production of superoxide andother ROS in the cells (29,30). Treating cells with antimycin-A resultedin decreased ROS levels in maspin overexpressing cells (p<0.05; FIG.2C). Additionally, hydroethidine (HE) was used to determine thesuperoxide (O2°-) levels as an indicator of oxidative stress in responseto an alternative ROS inducer i.e. staurosporine (STS) using HPLC-MS inTM40DNeo and TM40DMpcells. FIG. 2D depicts the schematic representationof the reaction of HE and superoxide radical anion (O2°-) to form2-hydroxyethidine (2-OH-E+). We observed a significant decrease in thelevels of superoxide in maspin overexpressing cells as compared toTM40DNeo cells in response STS treatment (p<0.05; FIG. 2E).

Cysteine residues in maspin control oxidative stress—Having establishedthat maspin is involved in modulating oxidative stress in normal mammaryepithelial and cancer cells, we further sought to identify theunderlying mechanism of maspin action. Due to presence of active thiol[—SH] group, cysteine residues play an important role as oxidativestress sensors in various proteins (12,13). Using Molsoft ICM-proVersion 3.48, we analyzed the X-ray structure of human maspin proteinreported by Law et al. (PDB ID: 1xu8) (16). We identified that out ofeight cysteine residues present in human maspin only three residues atthe position 183, 205, and 323 were structurally exposed, which mightserve as possible sites for oxidation. In contrast, cysteine residues atposition 20, 34, 214, 287, 373 were found to be buried. Previously, ithas been shown that mutating all the cysteines to serine/alanine by sitedirected mutagenesis does not change the structure of maspin (17).Therefore, we mutated either single (C183 or C323) or all the threeexposed cysteine residues to serine. FIG. 3A depicts three-dimensionalstructure of maspin demonstrating three structurally exposed (C183, C205and C323) cysteine residues. TM40D cells were transfected with differentmaspin cysteine mutants and expression of maspin was confirmed byWestern blot analysis (FIG. 3B).

Mutating a single cysteine residue i.e. 183 or 323 individually inmaspin led to a two-fold increase in ROS levels in comparison to TM40DMpcells (FIG. 3C); suggesting that both these cysteine residues areequally important in regulating cellular ROS. When all the three surfacecysteines were mutated, cellular ROS level in TM40DT was furtherincreased (˜1.4 fold) compared with TM40DC183S or TM40DC323S cells(p<0.05; FIG. 3C). Increased ROS levels in TM40DT correlated with theabsence of surface-exposed cysteine residues, thereby establishing theimportance of maspin cysteine residues in modulating ROS.

Enzymes such as those involved in glutathione redox cycle play animportant role in detoxification of various oxidant species (31). Adecreased GSH/GSSG ratio is considered indicative of increased oxidativestress (32,33). We found that overexpression of maspin in TM40D cellsresulted in an increased ratio of GSH/GSSG (p<0.01; FIG. 3D). However,expressing triple mutant maspin in TM40D cells resulted in a decreasedratio of GSH/GSSG as compared with cells expressing WT maspin in theabsence of ROS stimulus (p<0.01; FIG. 3D).

Triple mutated maspin is biologically active and evidence for oxidationof cysteine thiol to sulfenic acid—To confirm that mutated maspinretains its normal biological activity we tested whether the triplemutated recombinant maspin (GST.MpT) could increase cell adhesion to ECMmatrix as similar to WT maspin protein (GST.MpWT). Increased adhesion ofTM40DNeo cells to matrigel matrix was observed when cells werepretreated with GST.MpT compared to its control (p<0.01; FIG. 4A). Inorder to elucidate whether selected cysteine residues of maspin trapsfree radicals or other oxidants, WT and triple mutated recombinantmaspin proteins were treated with hydrogen peroxide in presence orabsence of dimedone. Dimedone is a highly specific agent that reactswith cysteine sulfenic acid (14,15). Sulfenic acid is the initialoxidation product which subsequently reacts with dimedone to form astable covalent bond (15) (FIG. 4B). When probed with anti-cysteinesulfenic acid antibody, we observed an absence of signal in lanes loadedwith triple mutated maspin as compared to WT maspin (FIG. 4C). Theseresults clearly demonstrate oxidation of thiol groups to cysteinesulfenic acid in maspin.

Moreover, to prove the participation of cysteine residues in maintainingthe redox state in cells, we immunoprecipitated maspin from hydrogenperoxide treated TM40DMp, TM40DC183S, TM40DC323S and TM40DTcells in thepresence or absence of dimedone and the IP-products were analyzed forthe presence of dimedone derivatized cysteine sulfenic acid residuesunder non-denaturing conditions. FIG. 4D shows the presence of maspincysteine sulfenic acid in TM40DMp, TM40DC183S and TM40DC323S cells butnot in the TM40DT cells. These results further demonstrate importance ofstructurally exposed cysteine residues. Similar results were obtainedwhen the experiment was performed with human mammary cancer epithelialMCF-7 cells (FIG. 4D, bottom panel), which constitutively expressesmaspin.

Cysteine residues affect cell proliferation pattern of TM40D cells viaactivation of ERK1/2—Previously, we and others have shown that maspinsuppresses tumor growth, invasion and metastasis of breast and prostatecancer (35, 36). To determine the effect of overexpression of maspin andcysteine mutations on the tumorigenic properties of TM40D cells, weassessed the anchorage independent growth and in vitro proliferation inmaspin WT and mutant TM40D cells using soft agar colony formation andMTT assays respectively. Anchorage-independent growth is one of thehallmarks of transformation and soft agar assay is a commonly used invitro assay for detecting malignant transformation of cells (28,37) Theabilities of the cells to form colonies on soft agar are shown in FIG.5A. Compared with the vector transfected cells, maspin overexpressedTM40DMp cells formed significantly fewer colonies/well (p<0.01; FIG.5A), a feature indicative of decrease in anchorage-independent growth,whereas no significant difference was observed when vector control wascompared with the triple mutated maspin (p>0.05). A significant increasewas observed in the colony formation between TM40DMp and TM40DT cells(p<0.01). Results of MTT proliferation assay revealed that WT maspinoverexpressing TM40DMp cells have low proliferation rate when comparedwith TM40DNeo and TM40DT cells over a time period of 96 hrs (FIG. 5B).To obtain direct evidence that deviation in proliferation pattern inTM40DNeo and TM40DT cells is due to changed ROS levels, we treated bothtypes of cells with N-acetyl cysteine (NAC; a strong antioxidant). Weobserved a significant drop in proliferation rate of TM40DNeo and TM40DTcells with the NAC treatment, whereas, no significant change wasobserved in TM40DMp cells (FIG. 5C). Thus these results provide strongevidence linking a high proliferation rate to elevated intracellular ROSlevels. Increased oxidative stress has been shown to cause proliferationdefects in cells via targeting key signaling molecules like ERK1/2 (38).The total ERK1/2 MAP kinase level was found to be nearly constant in allthree cell lines derived from TM40D by Western blot analysis (FIG. 5D).However, the corresponding phosphorylated activated forms (p-ERK1/2)were found to be consistently increased in TM40DNeo and TM40DT cells ascompared to TM40DMp cells suggesting that TM40DNeo and TM40DT cells havehigher constitutive expression of activated pERK1/2 than TM40DMp cells(FIG. 5D). Treating cells with the anti-oxidant NAC significantlyreduced the levels of activated pERK1/2 (FIG. 5E) and cell proliferationrate in TM40DNeo and TM40DT cells, however, no change was observed inTM40DMp. These results, clearly demonstrate that maspin inhibits ERK1/2activities, likely through ROS scavenging and this mechanism controlsthe epithelial cell proliferation.

Discussion

Oxidative damage to any cellular constituent, if remained unchecked, canlead to disease development (1, 39). Maspin belongs to the serpin familyof non-inhibitory protease inhibitor (3) and is abundantly produced innormal mammary luminal epithelial and myoepithelial cells (3, 40). Ourlaboratory and others have demonstrated maspin acts as a multifacetedprotein, interacting with extra- and intracellular groups of proteinsand regulating key functions of cell adhesion, motility, apoptosis, andangiogenesis. It is also critical in mammary gland development (4, 18,41). Role of maspin in oxidative stress has been speculated in the lastfew years (9, 11). In the present study, we provide evidence foroxidation of cysteine residues in maspin for the first time.Additionally, we demonstrate that maspin acts as a ROS scavenger toprovide resistance against oxidative stress. This suggests a newparadigm that maspin allows tumor cells to proliferate in an environmentof oxidative stress by maintaining redox homeostasis. Our data alsoshows that surface-exposed cysteines regulate ROS as an intracellularserpin. Results of adhesion assay (FIG. 4A) clearly demonstrate thatthis function of ROS regulation is independent of maspin's extracellularfunction, which increases cell adhesion to ECM matrix when maspin issecreted.

Our results (FIG. 1) indicate that cells expressing maspin have lowlevels of ROS due to the active participation of maspin in scavengingoxidants. The coordinated action of various cellular anti-oxidants inmammalian cells is critical for maintaining a steady redox state. Also,evidence suggests that maspin expression is regulated in response toredox status. Overexpression of MnSOD leads to an increased maspinexpression (42,43). This reinforces involvement of maspin in theregulation of cellular redox homeostasis.

We tested whether maspin expression level in normal mammary epithelialcells or in cells with reduced level of maspin (e.g. MCF-10A229)regulates cellular redox status. We then reintroduced maspin into tumorcells that do not express maspin, to study the mechanism ofmaspin-mediated regulation of cellular ROS. A reduction in totalintracellular ROS (FIG. 2B) in maspin overexpressing mouse mammaryepithelial cell (TM40D) further substantiates the importance of maspinin maintaining the redox state of cells. Increasing ROS in themitochondria (antimycin-A treatment) shows the physiological relevance(FIG. 2C) of maspin in oxidative stress, which is consistent with ourfinding that maspin attenuates ROS species such as superoxide (O2°-) inSTS-stressed TM40D cells (FIG. 2E). Taken together, our results indicatethat presence of maspin in mammary cells make them more efficient tofight against ROS irrespective of their origins; whether they arederived from mouse tumor epithelial cell (TM40D), or primary mouseepithelial cells, or human mammary epithelial MCF-10A cells. Emergingevidence suggests that redox-sensitive cysteine residues in proteins mayfunction as an oxidant sensor (44,45). The conversion of these residuesto sulfenic acid has been demonstrated for redox signaling in yeast,T-cell activation, and in other proteins (14,15,45), providing strongsupport for the growing roles of this modification in biology. Mutatingthree cysteine residues in maspin resulted in significant increase inintracellular ROS levels in TM40DT cells (FIG. 3C). Further, levels ofother anti-oxidant proteins were also found to alter in cells expressingcysteine to serine mutant maspin (TM40DT) when compared with the WTmaspin expressing cells (TM40DMp). Our data suggests that thesecysteines have a cumulative effect because mutation of either C183S orC323S led to similar significant increase in cellular ROS level. Thecells with triple mutations in maspin had ˜1.4 fold increase in ROSlevel than cells with single cysteine mutation (FIG. 3C). However, whilenot limiting the present invention, it is possible that certain redoxagents may preferentially attack a particular cysteine on maspin in vivodepending on their size and structure.

Besides its importance in redox homeostasis, overexpression of maspinalso inhibits cancer cell proliferation. Maspin overexpressing cellshave reduced capacities to form colonies and to grow in soft agar.Mutation of cysteine residues resulted in a phenotype, which forms morecolonies (FIG. 5A). Results of MTT assay revealed that TM40DNeo cellsproliferate more rapidly than WT maspin overexpressing cells. Increasedproliferation of TM40DNeo cells coincides with the increased ROS levelsin these cells (FIGS. 2B and 5B). Cells with mutated maspin (TM40DT)also proliferate more rapidly when compared to TM40DMp cells (FIG. 5B).In agreement with this, Cia et al have also reported decreasedproliferation rate with the maspin overexpression in esophagealcarcinoma (46). Also, NAC treatment reversed the oxidative stressphenotype of the maspin deficient or mutated maspin cells and thusindicating proliferation was a result of increased ROS in these cells.ROS can activate kinases and/or inhibit phosphatases resulting instimulation of signaling pathways such as ERK1/2 (mitogen activatedprotein kinases) that are important in cell proliferation,differentiation, invasion, and apoptosis (47). In the present study, wefound higher levels of phospho-ERK1/2 in TM40DNeo and TM40DT cells ascompared with TM40DMp cells (FIG. 5D). Anti-oxidant pre-treatment led toa decrease in expression of pERK1/2 in these cells (FIG. 5E). Theseobservations suggest that an increased oxidative stress activatesERK1/2, which in turn increases proliferation. These resultscollectively substantiate that maspin overexpressing cells have lowerROS and therefore maintain lower levels of pERK1/2 and proliferationrates, as compared with cell lines that have attenuated or mutatedmaspin expression.

This study indicates that under oxidative stress, cysteine thiols [—SH]in maspin are oxidized to cysteine sulfenic acid [—SOH], which mayfurther interact with GST (9) or some other unidentified molecule(s)which reduces the oxidized thiol in the cells (e.g. glutathione,peroxiredoxin etc). Reducing—SOH to —SH in maspin makes it available forrecycling and for scavenging more ROS in the intracellularmicroenvironment. Increased oxidative stress in cells with triplemutated maspin demonstrates the absence of above proposed mechanism.Cells expressing WT maspin have low proliferation rate, which isattributed to the low ROS levels, and therefore, suppressed tumorgrowth. In conclusion, maspin overexpression leads to resistance againstoxidative stress and maspin cysteine residues play an important role inmaintaining the redox status of cells.

Example 2 Maspin Interacts with Trx-1 in Redox Regulation

Maspin displays a conformational change from open to closed statusaround the G-helix region (Law et al., J Biol Chem (2005), hereinincorporated by reference). Three structurally close cysteines in maspinprotein (C20, and C287 or C373) are exposed in opened conformation,which are subject to ROS attacks and form disulfide bond within maspinmolecule. In fact, we had previously observed the formation of maspinintramolecular disulfide bonds in human breast epithelial MCF10A cells(Nawata, et al., Int J Mol Med 27, 249-54 (2011), herein incorporated byreference). In biological systems, cytoplasmic disulfide bonds aregenerally taken care of by Trx1 (Nakamura et al, Antioxid Redox Signal7, 823-8 (2005), herein incorporated by reference). To determine whethermaspin disulfide bond interacts with Trx1, we first transfectedflag-tagged cysteine mutated Trx1 (cysteine C₃₅ is mutated to alanine)to the maspin-expressing MCF7 breast tumor cells and established stabletransfectants. The mutant Trx1-C35A allows us to probe maspin-Trx1interaction because the intermediate complex between maspin and theTrx1-C35A is stable in cells. We exposed stable cloned cells with H₂O₂at 250 μM for 3 hours, harvested cell extracts for immunoprecipitationwith anti-flag antibody, and followed with Western blot analysis usinganti-maspin antibody. Our data showed that maspin indeed interacts withTrx1 in MCF-7 cells (FIG. 6). Recently, we identified cysteine 20 as thekey residue involved in maspin interaction with Trx1. It forms adisulfide bond with the adjacent cysteine 287 or cysteine 373, promotingmaspin protein to undergo a structural change to the closed conformationupon ROS attack. The closed conformation will likely change back to theopen conformation through reduction reaction with Trx1. This newdiscovery, together with our previous findings that maspin uses itsthree cysteines to control ROS, explain why maspin is such a powerfulanti-ROS scavenger against inflammation, cancer, and aging in variouscells and tissues.

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All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A method of treating an inflammatory condition and/or skinaging comprising: administering to a subject a composition comprisingmaspin, a maspin derivative, or a maspin mimetic.
 2. The method of claim1, wherein said administering is to the skin of said subject.
 3. Themethod of claim 2, wherein said composition further comprises skinlotion, cream, gel, or an ointment, a spray, or is present in a skinpatch.
 4. The method of claim 1, wherein said maspin derivativecomprises an amino acid sequence selected from SEQ ID NOs:1-4.
 5. Themethod of claim 1, wherein said maspin derivative comprises an aminoacid sequence selected from SEQ ID NOs:5-7.
 6. The method of claim 1,wherein said maspin derivative comprises the amino acid sequence in SEQID NO:8.
 7. The method of claim 6, wherein said maspin derivativefurther comprises a peptide amphiphile.
 8. The method of claim 1,wherein said maspin derivative comprises an amino acid sequence selectedfrom SEQ ID NOs:9-12.
 9. The method of claim 1, wherein said maspin isfull length or at least 99% of full-length human maspin.
 10. The methodof claim 1, wherein said subject has, or is suspected of having,symptoms of an inflammatory condition selected from the group consistingof: benign prostate hyperplasia (BPH), inflammatory bowel disease (IBD),ROS-induced obesity, diabetes, and atherosclerosis.
 11. The method ofclaim 10, wherein said administering reduces or eliminates at least oneof said symptoms of said inflammatory condition.
 12. A compositioncomprising: i) a human skin moisturizing lotion, get, cream, orointment, and ii) maspin, a maspin derivative, or a maspin mimetic. 13.The composition of claim 12, wherein said maspin derivative comprises anamino acid sequence selected from SEQ ID NOs:1-4.
 14. The composition ofclaim 12, wherein said maspin derivative comprises an amino acidsequence selected from SEQ ID NOs:5-7.
 15. The composition of claim 12,wherein said maspin derivative comprises the amino acid sequence in SEQID NO:8.
 16. The composition of claim 15, wherein said maspin derivativefurther comprises a peptide amphiphile.
 17. The composition of claim 12,wherein said maspin derivative comprises an amino acid sequence selectedfrom SEQ ID NOs:9-12.
 18. The composition of claim 12, wherein saidmaspin is full length or at least 99% of full-length human maspin.
 19. Asystem comprising: i) a human skin moisturizing lotion, cream, gel, orointment, and ii) maspin, a maspin derivative, or a maspin mimetic. 20.The system of claim 19, wherein said maspin derivative comprise an aminoacid sequence selected from SEQ ID NOs:1-13.